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Oncogenic fusion proteins expressed in immature hematopoietic cells fail to recapitulate the transcriptional changes observed in human AML.

Rapin N, Porse BT - Oncogenesis (2014)

Bottom Line: Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach.Surprisingly, we found that the gene-expression profiles of CD34+ human HSPCs transformed with the potent oncogenic fusion proteins AML-ETO or MLL-AF9, only weakly resembled those derived from primary AML samples.Hence, our work raises concerns as to the relevance of the use of in vitro transduced cells to study the impact of transcriptional deregulation in human AML.

View Article: PubMed Central - PubMed

Affiliation: 1] The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark [2] Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark [3] Department of Biology, Faculty of Natural Sciences, The Bioinformatics Centre, University of Copenhagen, Copenhagen, Denmark [4] Danish Stem Cell Centre (DanStem) Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
Reciprocal chromosomal translocations are observed in one-third of acute myeloid leukemia (AML) cases. Targeting and understanding the effects of the resulting aberrant oncogenic fusion proteins may help developing drugs against specific leukemic subtypes, as demonstrated earlier by the use of ATRA in acute promyelocytic leukemia. Hematopoietic stem/progenitor (HSPCs) cells transduced with oncogenic fusion genes are regarded as promising in vitromodels of their corresponding AML subtypes. Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach. Surprisingly, we found that the gene-expression profiles of CD34+ human HSPCs transformed with the potent oncogenic fusion proteins AML-ETO or MLL-AF9, only weakly resembled those derived from primary AML samples. Hence, our work raises concerns as to the relevance of the use of in vitro transduced cells to study the impact of transcriptional deregulation in human AML.

No MeSH data available.


Related in: MedlinePlus

Side-by-side comparison of gene expression profiles derived from AML blasts and fusion gene-transduced CD34+ cells cultured in vitro. (a) Mapping of relevant samples into the PCA space of the hierarchy of normal hematopoiesis. The replicates of the different populations have been averaged into one data point for readability. Hematopoietic stem cells (HSCs); multi-potent progenitors (MPPs); common myeloid progenitors (CMPs); granulocyte–monocyte progenitors (GMPs); megakaryocyte–erythrocyte progenitors (MEPs); early promyelocytes (early PMs); late promyelocytes (late PMs); myelocytes (MYs); metamyelocytes (MMs); band cells (BCs); polymorphonuclear neutrophilic granulocytes (PMN_BM); monocytes (Mono); empty vector control CD34+ cells at 6 h (c_6 h), 3 days (c_3 d) and 8 days (c_8 d); MLL-AF9-expressing CD34+ cells at 6 h (mll_6 h), 3 days (mll_3 d) and 8 days (mll_8 d); AML-1ETO-expressing CD34+ cells at 6 h (eto_6 h), 3 days (eto_3 d) and 8 days (eto_8 d); leukemic blasts from patients with t(8;21) AML (AML with t(8;21)); leukemic blasts from patients with MLL-rearranged AML (AML with t(11q23)/MLL). The PCA was performed on 2119 probe sets selected by variance filtering.16 (b) Stem cell score of gene expression profiles of transformed cells, AML blasts and normal HSPCs. (c) Hierarchical clustering of samples in (a) using genes from the gene signatures RAPIN_CVN_t(8;21)_up/_dn and RAPIN_CVN_t(11q23)_MLL_up/_dn.16 (d) AML1-ETO- and MLL-published gene signatures enrichment represented as –log(P-value) for transformed cells and AML blasts (MLL signatures *P<0.05, **P<0.001, ***P<1e5; AML1-ETO signatures °P<0.05, °°P<0.001, °°°P<1e5). (e) Overlap between genes deregulated (/log2-fold change/>1, P<5e−3, moderated t-test) in AML with t(8;21) versus normal cells and AML1-ETO-transduced CD34+ cells versus control after 8 days of culture. (f) Overlap between genes deregulated (/log2-fold change/>1, P<5e−3, moderated t-test) in AML with t(11q23)/MLL versus normal cells and MLL-AF9-transduced CD34+ cells versus control after 8 days of culture. (g) Correlation between the extent of deregulation in AML with t(8;21) and transduced CD34+ cells of the genes selected in e. Genes displaying good correlation (AML blast fold change=transduced CD34+ cells fold change±0.25) are depicted. (h) Same as g for MLL-rearranged AML using genes selected in f.
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fig1: Side-by-side comparison of gene expression profiles derived from AML blasts and fusion gene-transduced CD34+ cells cultured in vitro. (a) Mapping of relevant samples into the PCA space of the hierarchy of normal hematopoiesis. The replicates of the different populations have been averaged into one data point for readability. Hematopoietic stem cells (HSCs); multi-potent progenitors (MPPs); common myeloid progenitors (CMPs); granulocyte–monocyte progenitors (GMPs); megakaryocyte–erythrocyte progenitors (MEPs); early promyelocytes (early PMs); late promyelocytes (late PMs); myelocytes (MYs); metamyelocytes (MMs); band cells (BCs); polymorphonuclear neutrophilic granulocytes (PMN_BM); monocytes (Mono); empty vector control CD34+ cells at 6 h (c_6 h), 3 days (c_3 d) and 8 days (c_8 d); MLL-AF9-expressing CD34+ cells at 6 h (mll_6 h), 3 days (mll_3 d) and 8 days (mll_8 d); AML-1ETO-expressing CD34+ cells at 6 h (eto_6 h), 3 days (eto_3 d) and 8 days (eto_8 d); leukemic blasts from patients with t(8;21) AML (AML with t(8;21)); leukemic blasts from patients with MLL-rearranged AML (AML with t(11q23)/MLL). The PCA was performed on 2119 probe sets selected by variance filtering.16 (b) Stem cell score of gene expression profiles of transformed cells, AML blasts and normal HSPCs. (c) Hierarchical clustering of samples in (a) using genes from the gene signatures RAPIN_CVN_t(8;21)_up/_dn and RAPIN_CVN_t(11q23)_MLL_up/_dn.16 (d) AML1-ETO- and MLL-published gene signatures enrichment represented as –log(P-value) for transformed cells and AML blasts (MLL signatures *P<0.05, **P<0.001, ***P<1e5; AML1-ETO signatures °P<0.05, °°P<0.001, °°°P<1e5). (e) Overlap between genes deregulated (/log2-fold change/>1, P<5e−3, moderated t-test) in AML with t(8;21) versus normal cells and AML1-ETO-transduced CD34+ cells versus control after 8 days of culture. (f) Overlap between genes deregulated (/log2-fold change/>1, P<5e−3, moderated t-test) in AML with t(11q23)/MLL versus normal cells and MLL-AF9-transduced CD34+ cells versus control after 8 days of culture. (g) Correlation between the extent of deregulation in AML with t(8;21) and transduced CD34+ cells of the genes selected in e. Genes displaying good correlation (AML blast fold change=transduced CD34+ cells fold change±0.25) are depicted. (h) Same as g for MLL-rearranged AML using genes selected in f.

Mentions: In order to determine the extent to which fusion protein-expressing HSPCs cultured in vitro mirrored the transcriptional changes observed in primary leukemic blasts, we collected microarray-based gene expression data from several sources (Table 1). These include normal HSPCs, empty vector-, MLL-AF9- and AML-ETO-transduced CD34+ cells cultured in vitro (6 h, 3 d or 8 d after transduction) as well as primary leukemic blast from patients with corresponding karyotypic lesions. Using our recent cancer versus normal (CvN) approach based on principal component analysis (PCA), we mapped gene expression profiles from in vitro cultured cells and patient samples onto the gene expression landscape of normal hematopoiesis (Figure 1a).16 Strikingly, we find that the transduced cells cluster tightly as a function of time but independent of the expression of the transforming oncogene. Moreover, the oncogene-transduced CD34+ cells map nowhere near their respective patient counterparts. Therefore, these findings suggest that the main driver of the transcriptional changes of transduced cells is related to the culturing process and not to the expression of the oncogenic fusion protein. Indeed, when we quantify the extent of differentiation using a newly defined ‘stemness' score, we find that the transduced cells progressively lose stemness (that is, they differentiate) over time in a manner independent of the expression of the fusion oncogene (Figure 1b).


Oncogenic fusion proteins expressed in immature hematopoietic cells fail to recapitulate the transcriptional changes observed in human AML.

Rapin N, Porse BT - Oncogenesis (2014)

Side-by-side comparison of gene expression profiles derived from AML blasts and fusion gene-transduced CD34+ cells cultured in vitro. (a) Mapping of relevant samples into the PCA space of the hierarchy of normal hematopoiesis. The replicates of the different populations have been averaged into one data point for readability. Hematopoietic stem cells (HSCs); multi-potent progenitors (MPPs); common myeloid progenitors (CMPs); granulocyte–monocyte progenitors (GMPs); megakaryocyte–erythrocyte progenitors (MEPs); early promyelocytes (early PMs); late promyelocytes (late PMs); myelocytes (MYs); metamyelocytes (MMs); band cells (BCs); polymorphonuclear neutrophilic granulocytes (PMN_BM); monocytes (Mono); empty vector control CD34+ cells at 6 h (c_6 h), 3 days (c_3 d) and 8 days (c_8 d); MLL-AF9-expressing CD34+ cells at 6 h (mll_6 h), 3 days (mll_3 d) and 8 days (mll_8 d); AML-1ETO-expressing CD34+ cells at 6 h (eto_6 h), 3 days (eto_3 d) and 8 days (eto_8 d); leukemic blasts from patients with t(8;21) AML (AML with t(8;21)); leukemic blasts from patients with MLL-rearranged AML (AML with t(11q23)/MLL). The PCA was performed on 2119 probe sets selected by variance filtering.16 (b) Stem cell score of gene expression profiles of transformed cells, AML blasts and normal HSPCs. (c) Hierarchical clustering of samples in (a) using genes from the gene signatures RAPIN_CVN_t(8;21)_up/_dn and RAPIN_CVN_t(11q23)_MLL_up/_dn.16 (d) AML1-ETO- and MLL-published gene signatures enrichment represented as –log(P-value) for transformed cells and AML blasts (MLL signatures *P<0.05, **P<0.001, ***P<1e5; AML1-ETO signatures °P<0.05, °°P<0.001, °°°P<1e5). (e) Overlap between genes deregulated (/log2-fold change/>1, P<5e−3, moderated t-test) in AML with t(8;21) versus normal cells and AML1-ETO-transduced CD34+ cells versus control after 8 days of culture. (f) Overlap between genes deregulated (/log2-fold change/>1, P<5e−3, moderated t-test) in AML with t(11q23)/MLL versus normal cells and MLL-AF9-transduced CD34+ cells versus control after 8 days of culture. (g) Correlation between the extent of deregulation in AML with t(8;21) and transduced CD34+ cells of the genes selected in e. Genes displaying good correlation (AML blast fold change=transduced CD34+ cells fold change±0.25) are depicted. (h) Same as g for MLL-rearranged AML using genes selected in f.
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Related In: Results  -  Collection

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fig1: Side-by-side comparison of gene expression profiles derived from AML blasts and fusion gene-transduced CD34+ cells cultured in vitro. (a) Mapping of relevant samples into the PCA space of the hierarchy of normal hematopoiesis. The replicates of the different populations have been averaged into one data point for readability. Hematopoietic stem cells (HSCs); multi-potent progenitors (MPPs); common myeloid progenitors (CMPs); granulocyte–monocyte progenitors (GMPs); megakaryocyte–erythrocyte progenitors (MEPs); early promyelocytes (early PMs); late promyelocytes (late PMs); myelocytes (MYs); metamyelocytes (MMs); band cells (BCs); polymorphonuclear neutrophilic granulocytes (PMN_BM); monocytes (Mono); empty vector control CD34+ cells at 6 h (c_6 h), 3 days (c_3 d) and 8 days (c_8 d); MLL-AF9-expressing CD34+ cells at 6 h (mll_6 h), 3 days (mll_3 d) and 8 days (mll_8 d); AML-1ETO-expressing CD34+ cells at 6 h (eto_6 h), 3 days (eto_3 d) and 8 days (eto_8 d); leukemic blasts from patients with t(8;21) AML (AML with t(8;21)); leukemic blasts from patients with MLL-rearranged AML (AML with t(11q23)/MLL). The PCA was performed on 2119 probe sets selected by variance filtering.16 (b) Stem cell score of gene expression profiles of transformed cells, AML blasts and normal HSPCs. (c) Hierarchical clustering of samples in (a) using genes from the gene signatures RAPIN_CVN_t(8;21)_up/_dn and RAPIN_CVN_t(11q23)_MLL_up/_dn.16 (d) AML1-ETO- and MLL-published gene signatures enrichment represented as –log(P-value) for transformed cells and AML blasts (MLL signatures *P<0.05, **P<0.001, ***P<1e5; AML1-ETO signatures °P<0.05, °°P<0.001, °°°P<1e5). (e) Overlap between genes deregulated (/log2-fold change/>1, P<5e−3, moderated t-test) in AML with t(8;21) versus normal cells and AML1-ETO-transduced CD34+ cells versus control after 8 days of culture. (f) Overlap between genes deregulated (/log2-fold change/>1, P<5e−3, moderated t-test) in AML with t(11q23)/MLL versus normal cells and MLL-AF9-transduced CD34+ cells versus control after 8 days of culture. (g) Correlation between the extent of deregulation in AML with t(8;21) and transduced CD34+ cells of the genes selected in e. Genes displaying good correlation (AML blast fold change=transduced CD34+ cells fold change±0.25) are depicted. (h) Same as g for MLL-rearranged AML using genes selected in f.
Mentions: In order to determine the extent to which fusion protein-expressing HSPCs cultured in vitro mirrored the transcriptional changes observed in primary leukemic blasts, we collected microarray-based gene expression data from several sources (Table 1). These include normal HSPCs, empty vector-, MLL-AF9- and AML-ETO-transduced CD34+ cells cultured in vitro (6 h, 3 d or 8 d after transduction) as well as primary leukemic blast from patients with corresponding karyotypic lesions. Using our recent cancer versus normal (CvN) approach based on principal component analysis (PCA), we mapped gene expression profiles from in vitro cultured cells and patient samples onto the gene expression landscape of normal hematopoiesis (Figure 1a).16 Strikingly, we find that the transduced cells cluster tightly as a function of time but independent of the expression of the transforming oncogene. Moreover, the oncogene-transduced CD34+ cells map nowhere near their respective patient counterparts. Therefore, these findings suggest that the main driver of the transcriptional changes of transduced cells is related to the culturing process and not to the expression of the oncogenic fusion protein. Indeed, when we quantify the extent of differentiation using a newly defined ‘stemness' score, we find that the transduced cells progressively lose stemness (that is, they differentiate) over time in a manner independent of the expression of the fusion oncogene (Figure 1b).

Bottom Line: Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach.Surprisingly, we found that the gene-expression profiles of CD34+ human HSPCs transformed with the potent oncogenic fusion proteins AML-ETO or MLL-AF9, only weakly resembled those derived from primary AML samples.Hence, our work raises concerns as to the relevance of the use of in vitro transduced cells to study the impact of transcriptional deregulation in human AML.

View Article: PubMed Central - PubMed

Affiliation: 1] The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark [2] Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark [3] Department of Biology, Faculty of Natural Sciences, The Bioinformatics Centre, University of Copenhagen, Copenhagen, Denmark [4] Danish Stem Cell Centre (DanStem) Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
Reciprocal chromosomal translocations are observed in one-third of acute myeloid leukemia (AML) cases. Targeting and understanding the effects of the resulting aberrant oncogenic fusion proteins may help developing drugs against specific leukemic subtypes, as demonstrated earlier by the use of ATRA in acute promyelocytic leukemia. Hematopoietic stem/progenitor (HSPCs) cells transduced with oncogenic fusion genes are regarded as promising in vitromodels of their corresponding AML subtypes. Here, we critically assessed the potential of such in vitro models using an integrative bioinformatics approach. Surprisingly, we found that the gene-expression profiles of CD34+ human HSPCs transformed with the potent oncogenic fusion proteins AML-ETO or MLL-AF9, only weakly resembled those derived from primary AML samples. Hence, our work raises concerns as to the relevance of the use of in vitro transduced cells to study the impact of transcriptional deregulation in human AML.

No MeSH data available.


Related in: MedlinePlus